Fig. 2: ScoC induces DNA looping.

A FRET analysis demonstrating DNA looping mediated by ScoC. The fluorescence intensity was measured at 30 °C in the presence of different concentrations of ScoC with 10 nM of a 5’-Cy3 3’-Cy5 labeled 248-bp DNA element. The element was amplified from the oppA operator region and contained the two ScoC-binding sites at the ends. On the right is a cartoon demonstrating the equilibrium between the different intermediates, where D stands for DNA, and P stands for the tetrameric ScoC structure. At ScoC concentrations above 10 nM the equilibrium is shifted towards the DPP form, resulting in reduced fluorescence intensity. B Electrophoretic mobility shift assays demonstrating different DNA forms obtained upon binding of ScoC. Left panel: ScoC was incubated with a fluorescently labeled 316-bp DNA fragment containing two ScoC-binding sites at its ends. Two distinct retarded bands can be visualized. A slow migrating band representing DNA in the loop form (DP*) and a faster migrating band, representing the DNA in its linear form, in which two independent ScoC tetramers are bound at each end (DPP). In higher concentrations of ScoC only the fast-running band of the linear form appears. The DP form is not visible presumably since the equilibrium (K₂) tends strongly towards the loop form (DP*). Right panel: a 247-bp DNA containing a single ScoC-binding site gives only a single retarded band shift representing a linear DNA form with one ScoC tetramer bound. Numerical source data are in Supplementary Data 2. C Visualizing ScoC-induced DNA looping by atomic force microscopy. AFM topography images of A 316 bp DNA fragment, B ScoC, C ScoC-DNA mixture showing different types of interactions and, D a zoom into a DNA loop induced by ScoC (yellow dashed square).